Androstane and pregnane steroids with potent allosteric gaba receptor chloride ionophore modulating properties

ABSTRACT

This invention describes compounds of Structures 1, 2, and 3 and their use as allosteric modulators of the GABA receptor chloride ionophore complex to alleviate stress, anxiety, mood disorders, seizures, depression, treatment of drug and alcohol abuse, memory, premenstrual disorders, and neural system damage.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 13/974,633,filed Aug. 23, 2013, now allowed, which is a divisional of U.S. Ser. No.12/664,470, filed May 5, 2010, now U.S. Pat. No. 8,575,375, which is a371 application of PCT/US08/67059, filed Jun. 16, 2008, and claimspriority to U.S. Provisional Ser. No. 60/944,257, filed Jun. 15, 2007.

BACKGROUND OF THE INVENTION

Field of Invention

The present invention relates to androstane and pregnane steroidcompounds and their use as allosteric modulators of the GABA receptorchloride ionophore complex and their use to alleviate stress, anxiety,mood disorders, seizures, depression, treatment of drug and alcoholabuse, memory, premenstrual disorders, and neural system damage.

Discussion of the Background

The present invention encompasses methods and compounds related toendogenous metabolites of androstane and pregnane steroids. Certainendogenous steroids including allopregnanolone(3α-hydroxy-5α-pregnane-20-one) and the A-ring reduced metabolite ofprogesterone are potent stereoselective positive allosteric modulatorsof GABA receptors (Belelli and Lambert, 2005). These neurosteroids, likeother agents that potentiate the activity of GABA receptors, exhibitanxiolytic, sedative-hypnotic, anticonvulsant, and general anestheticactions. Allopregnanolone and A-ring reduced neurosteroids generallylack classical hormonal activity mediated by nuclear hormone receptors.Given their GABA receptor modulating activity, natural neurosteroidscould potentially be used therapeutically (Gasior et al., 1999).However, they do not have ideal properties as drug substances. Naturalneurosteroids can be metabolically converted to hormonally activesubstances (Rupprecht, 2003) and have poor bioavailability.Consequently, there is an unmet need for synthetic analogs with improvedpharmacokinetic and pharmacodynamic properties. Structural modificationsat the 3-position inhibit metabolism of the secondary 3-hydroxylsubstituent, but permit GABA receptor modulating activity to be retained(Hogenkamp et al., 1997). One such analog, ganaxalone, the 3β-methylderivative of allopregnanolone, is currently in clinical development forepilepsy (Monaghan et al., 1999; Rogawski, 2006).

Substitution of the allopregnanolone 17-position with a variety offunctional groups allows GABA receptor modulating activity to beretained. For example, the naturally occurring neurosteroids,allotetrahydrodeoxycorticosterone (5α,3α-THDOC), androstenediol, andandrosterone, which have O═CCH₂OH, alcohol, and keto substituents at the17-position have GABA receptor modulating activity as does the prototypeallopregnanolone, which has an acetyl substituent at the 17-position. Inaddition, the synthetic 17β-carbonitrile analog exhibits GABA receptormodulatory potency and efficacy similar to allopregnanolone (Wittmer etal., 1996).

Interestingly, the natural (16-17 unsaturated) pheromone,3α-androstenol, lacks a 17-position substitutent yet retains GABAreceptor modulating activity, albeit of reduced potency (Kaminski etal., 2006). Taken collectively, these data indicate a critical role forGABA modulators based on the neuroactive steroid scaffold for treatmentof a variety of disease states.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIGS. 1A and 1B are graphical representations of the results obtainedwhen A-2a was evaluated in the elevated zero maze test of anxiolyticactivity. The percentage of time spent in open arms of the maze (FIG.1A) as well as the number of entries (FIG. 1B) were significantlyenhanced with therapeutic doses of A-2a.

FIG. 2 is a graphical representation showing the percent protectionprovided by A-2a, A-2b, and B-5 from pentylenetetrazole (PTZ) inducedseizures. Compounds exhibited potent anticonvulsant activity in the EC₅₀range of 5-13 nM.

FIG. 3 is a graphical representation of the examination of A-2a, in twodifferent seizure models demonstrating the ability of this syntheticneuroactive steroid to reduce both chemically (PTZ) and electrically (6Hz) induced seizures in mice.

FIG. 4 provides a synthetic scheme for the preparation of compoundsA1-A2.

FIG. 5 provides a synthetic scheme for the preparation of compoundsB1-B7.

FIG. 6 provides a synthetic scheme for the preparation of compoundsC1-C8.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide novelpregnane and androstane steroids having allosteric GABA activity.

A further object of the present invention is to provide methods forusing these novel compounds in the treatment of convulsions, epilepsy,depression, drug and alcohol abuse, anxiety, memory problems, and neuralsystem damage in humans or animals.

These and other objects of the present invention, either alone or incombinations thereof, have been satisfied by the discovery of steroidderivatives having formula 1, 2, or 3 below:

DETAILED DESCRIPTION OF THE INVENTION

The present invention expands upon the structural features on pregnaneand androstane steroids previously been found to be associated withallosteric GABA activity. It thus provides an array of compounds fromwhich may be obtained additional analogs with improved pharmacologicaland pharmacokinetic properties. Prior art describes the utility of the17β-acetyl substituent in steroidal 3α-ols for GABA activity. In thiswork we show that a 17β-nitro group or 17-thioalkyl group serve as abioisostere for 17β-acetyl and when combined with the 3α-OH-5α-H ofandrostane and pregnane steroid systems full agonist activity andequipotency with allopregnanolone in the [³H]-flunitrazepam assay isobserved for GABA activity. These derivatives are also equipotent withallopregnanolone in the [³⁵S]TBPS assay for GABA.

The effect on GABA allosteric potency via substitution at the11β-position with aromatic groups has not been previously examined forandrostane and pregnane steroids having a 3α-OH, 5α-H. In this work wenow demonstrate that substitution at the 11β-position with analternately substituted aromatic group provides androstane and pregnanesteroids with enhanced GABA allosteric potency compared toallopregnanolone in both the [³⁵S]FBPS assay for GABA and the[³H]-flunitrazepam assay. Novel structural features disclosed in thisapplication include the 17β-nitro group, 17β-thiomethyl group, and the11β-(4-dimethylaminophenyl) group. These functional groups will possessdifferent solubility, metabolism and pharmacokinetic properties from the17β-acetyl and 11β-H compounds of previous inventions. These may aid informulation or dosing. In addition, some of the compounds are excellentintermediates for further modifications. Such compounds may be of use inepilepsy, depression, anxiety, treatment of drug and alcohol abuse,memory, premenstrual disorders, neural system damage and otherpotentially therapeutic areas.

The steroid derivatives of this invention are those comprised of theabove mentioned structural formulas 1, 2, or 3.

Within the scope of the present invention, the term heteroatom meansoxygen, nitrogen, sulfur, silicon or boron. Halogen means fluorine,chlorine, bromine or iodine and halo means fluoro, chloro, bromo oriodo. Aralkyl, aralkenyl, or aralkynyl means a C₁-C₄ alkyl, C₂-C₄alkenyl or C₂-C₄ alkynyl group bearing an aryl substituent. Lower alkylmeans a C₁-C₆ alkyl group. Heteroaryl means a unit of 5 to 12non-hydrogen atoms consisting of one or more cyclic structures that maybe fused or linked together, which contain 1 to 5 heteroatoms and whichare generally accepted by those skilled in the art as having aromaticelectronic character.

Heteroaralkyl, heteroaralkenyl, or heteroaralkynyl means a C₁-C₄ alkyl,C₂-C₄ alkenyl, or C₂-C₄ alkynyl group bearing a heteroaryl substituent.

“Optionally substituted” means unsubstituted or substituted with one ormore heteroatom(s) and/or halogens and/or alkyl groups of 1 to 4 carbonatoms and/or alkenyl and/or alkynyl groups of 2 to 4 carbon atoms and/orcycloalkyl groups of 3 to 7 carbon atoms and/or aryl groups of 6 to 12carbon atoms and/or heteroaryl groups, and in which the alkyl, alkenyl,alkynyl, cycloalkyl, aryl or heteroaryl group may be further substitutedwith one or more heteroatoms and/or halogens. Substitution may occurdirectly on CH₂ groups of cyclic amine heterocycles. Where their valencypermits, heteroatoms may be substituted either within the carbon chainor by attachment to it by single or double bonds. For example,—CH₂CH₂C(═O)H, —CH₂(C═O)CH₃, —CH₂CH₂OCH₃, —CH₂CH₂CH₂OH, CH₃CH₂CH₂O—,CH₂CH₂C(═O)NH₂, CH₃CH₂C(═O)NH—, CH₂CH₂COOCH₃, CH₃CH₂COO—, and CF₃CC— allfall within this definition.

In all cases where valency and steric considerations permit, alkyl,alkenyl, alkynyl and cycloalkyl groups may contain additional double ortriple bonds and/or branched chains.

In one embodiment of the invention, the group R¹ as it appears instructure 1 may be H or (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, 2-arylsubstitutedethynyl, arylsubstituted C₁₋₄ alkyl, arylsubstituted C₂₋₄-alkenyl,arylsubstituted C₃₋₆ cycloalkyl, heterocycle; 2-heterocycle-substitutedethynyl, heterocycle-substituted C₁₋₄ alkyl, heterocycle-substitutedC₂₋₄-alkenyl, or heterocycle-substituted C₃₋₆ cycloalkyl;

the R² group is OH or OR¹⁴, where R¹⁴ is HCO— or (optionallysubstituted) C₁₋₁₈ alkyl-CO— (except that R¹⁴ is not CH₃ when R¹, R²,R³, R⁵, R⁶, R⁸, R⁹ and R¹⁰═H, R⁴═CH₃, and there are no double bondspresent), C₂₋₁₈ alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₆₋₁₀ aryl-CO— orheterocycle-CO—; or R¹⁴ is (optionally substituted) C₁₋₁₈ alkyl-X—CO—,C₂₋₁₈ alkenyl-X—CO—, C₂₋₁₈ alkynyl-X—CO—, C₆₋₁₀ aryl-X—CO— orheterocycle-X—CO—, where X is O or NR¹¹, where R¹¹ is (optionallysubstituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynylC₆₋₁₀ aryl or heterocycle; or R¹⁴ is trimethylsilyl or triethylsilyl; orR¹⁴ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄alkenyl, or C₂₋₄ alkynyl, or; or R¹⁴ is HO—SO₂— or a salt thereof or R²is NR¹⁵R¹⁶, where R¹⁵ is H, OH or (optionally substituted) C₁₋₄ alkyl,C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, heterocycle orR¹⁵ is OR¹⁷, where R¹⁷ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, or heterocycle,C₁₋₁₈ alkyl-CO—, C₂₋₁₈ alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₆₋₁₀ aryl-CO— orheterocycle-CO—; and where R¹⁶ is H or (optionally substituted) C₁₋₄alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, orheterocycle, or R¹⁶ is H—CO— or (optionally substituted) C₁₋₁₈alkyl-CO—, C₂₋₁₈ alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₃₋₆ cycloalkyl-CO—,C₆₋₁₀ aryl-CO— or heterocycle-CO—;

R³ is H, halogen, cyano, azido or (optionally substituted) C₁₋₄ alkyl,C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, or heterocycle;

R⁴ is H or (optionally substituted) Me;

R⁵ is H, halogen, azido, cyano, thiocyano, ═O, OH, OR¹⁶ (where R¹⁶ is asdefined above), or R⁵ is NH₂ or NR¹⁵R¹⁶ (where R¹⁵ and R¹⁶ are asdefined above); or R⁵ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, or C₂₋₄ alkenyl; or R⁵ is S(O)_(n)R¹⁶, whereR¹⁶ is as defined above and n=0, 1, or 2;

R⁶ is H, or (optionally substituted) C₁₋₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₈alkenyl, C₂₋₈ alkynyl, C₆₋₁₀ aryl or heterocycle;

R⁸ is H, halogen, azido, cyano, thiocyano, ═O, OH, OR¹⁶ (where R¹⁶ is asdefined above); or R⁵ is NH₂ or NR¹⁵R¹⁶ (where R¹⁵ and R¹⁶ are asdefined above); or R⁸ is (optionally substituted) C₁₋₁₈ alkyl, C₃₋₆cycloalkyl, C₂₋₁₈ alkynyl, C₂₋₁₈ alkenyl, C₆₋₁₀ aryl or heterocycle; orR⁸ is COOR¹⁸ where R¹⁸ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₆₋₁₀ aryl, or heterocycle; orR⁸ is S(O)R¹⁶, where R¹⁶ is as defined above and n=0, 1, or 2;

R⁹ is H, cyano, azido, halogen, thiocyano, OH, OR¹⁶, where R¹⁶ is asdescribed above, or R⁹ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, 2-arylsubstitutedethynyl, arylsubstituted C₁₋₄ alkyl, arylsubstituted C₂₋₄-alkenyl,arylsubstituted C₃₋₆ cycloalkyl, heterocycle; 2-heterocycle-substitutedethynyl, heterocycle-substituted C₁₋₄ alkyl, heterocycle-substitutedC₂₋₄-alkenyl, heterocycle-substituted C₃₋₆ cycloalkyl; and

R¹⁰ is H, keto, halogen, cyano, thiocyano, azido or NR¹⁵R¹⁶, where R¹⁵and R¹⁶ are as described above; or R¹⁰ is (optionally substituted) C₁₋₄alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl orheterocycle; or R¹⁰ is OR¹⁶ where R¹⁶ as described above or R¹⁶ istrimethylsilyl or triethylsilyl; or R¹⁶ is ═CH₂ or ═CHR¹⁹, where R¹⁹⁰ is(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl or C₆₋₁₀ aryl; or R¹⁰ is S(O)_(n)R¹⁶, where R¹⁶ is as definedabove and n=0, 1, or 2; and stereoisomers and pharmaceuticallyacceptable compositions thereof.

In another embodiment of the present invention, the group R¹ as itappears in structure 2 is H or (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, 2-arylsubstitutedethynyl, arylsubstituted C₁₋₄ alkyl, arylsubstituted C₂₋₄-alkenyl,arylsubstituted C₃₋₆ cycloalkyl, heterocycle, 2-heterocycle-substitutedethynyl, heterocycle-substituted C₁₋₄ alkyl, heterocycle-substitutedC₂₋₄-alkenyl, heterocycle-substituted C₃₋₆ cycloalkyl wherein m=0, 1, or2;

R² is OH or OR¹⁴, where R¹⁴ is HCO— or (optionally substituted) C₁₋₁₈alkyl-CO— (except that R¹⁴ is not CH₃ when R¹, R², R³, R⁵, R⁶, R⁸, R⁹and R¹⁰═H, R⁴═CH₃, and there are no double bonds present), C₂₋₁₈alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₆₋₁₀aryl-CO— or heterocycle-CO—; or R¹⁴is C₁₋₁₈ alkyl-X—CO—, C₂₋₁₈ alkenyl-X—CO—, C₂₋₁₈ alkynyl-X—CO—,aryl-X—CO— or heterocycle-X—CO—, where X is O or NH; or R¹⁴ istrimethylsilyl or triethylsilyl; or R¹⁴ is (optionally substituted) C₁₋₄alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, or C₂-4 alkenyl; or R¹⁴ is HO—SO₂—or a salt thereof; or R² is NR¹⁵R¹⁶, where R¹⁵ is H, OH or (optionallysubstituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl,C₆₋₁₀ aryl, heterocycle, or R¹⁵ is OR¹⁷, where R¹⁷ is (optionallysubstituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl,C₆₋₁₀ aryl, or heterocycle, C₁₋₁₈ alkyl-CO—, C₂₋₁₈ alkenyl-CO—, C₂₋₁₈alkynyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO—; and where R¹⁶ is H or(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl, C₆₋₁₀ aryl, or heterocycle, or R¹⁶ is H—CO— or (optionallysubstituted) C₁₋₁₈ alkyl-CO—, C₂₋₁₈ alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₃₋₆cycloalkyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO— or R¹⁶ is C₁₋₁₈alkyl-X—CO—, C₂₋₁₈ alkenyl-X—CO—, C₂₋₁₈ alkynyl-X—CO—, aryl-X—CO— orheterocycle-X—CO—, where X is O or NH; wherein m=0, 1, or 2;

R³ is H, halogen, cyano, azido or (optionally substituted) C₁₋₄ alkyl,C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, or heterocycle;wherein m=0, 1, or 2;

R⁴ is H or (optionally substituted) Me; wherein m=0, 1, or 2;

R⁵ is H, halogen, azido, cyano, thiocyano, ═O, OH, OR¹⁶ (where R¹⁶ is asdefined above), or R⁵ is NH₂ or NR¹⁵R¹⁶ (where R¹⁵ and R¹⁶ are asdefined above); or R⁵ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, or C₂₋₄ alkenyl; or R⁵ is S(O)R¹⁶, where R¹⁶is as defined above and n=0, 1, or 2; wherein m=0, 1, or 2;

R⁶ is H, or (optionally substituted) C₁₋₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₈alkenyl, C₂₋₈ alkynyl, C₆₋₁₀ aryl or heterocycle; wherein m=0, 1, or 2;

R⁷ is H, cyano or (optionally substituted) C₁₋₈ alkyl, C₃₋₆ cycloalkyl,C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₆₋₁₀ aryl or heterocycle; wherein m=0, 1,or 2;

R⁸ is H, halogen, azido, cyano, thiocyano, ═O, OH, OR¹⁶ (where R¹⁶ is asdefined above); or R⁵ is NH₂ or NR¹⁵R¹⁶ (where R¹⁵ and R¹⁶ are asdefined above); or R⁸ is (optionally substituted) C₁₋₁₈ alkyl, C₃₋₆cycloalkyl, C₂₋₁₈ alkynyl, C₂₋₁₈ alkenyl, C₆₋₁₀ aryl or heterocycle; orR⁸ is COOR¹⁸ where R¹⁸ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₆₋₁₀ aryl, or heterocycle; orR⁸ is S(O)R′⁶, where R¹⁶ is as defined above and n=0, 1, or 2; whereinm=0, 1, or 2;

R⁹ is H, cyano, azido, halogen, thiocyano, OH, OR¹⁶, where R¹⁶ is asdescribed above, or R⁹ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, 2-arylsubstitutedethynyl, arylsubstituted C₁₋₄ alkyl, arylsubstituted C₂₋₄-alkenyl,arylsubstituted C₃₋₆ cycloalkyl, heterocycle; 2-heterocycle-substitutedethynyl, heterocycle-substituted C₁₋₄ alkyl, heterocycle-substitutedC₂₋₄-alkenyl, heterocycle-substituted C₃₋₆ cycloalkyl; wherein m=0, 1,or 2; and

R¹⁰ is H, keto, halogen, cyano, thiocyano, azido or NR¹⁵R¹⁶, where R¹⁵and R¹⁶ are as described above; or R¹⁰ is (optionally substituted) C₁₋₄alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl orheterocycle; or R¹⁰ is OR¹⁶ where R¹⁶ as described above or R¹⁶ istrimethylsilyl or triethylsilyl; or R¹⁶ is ═CH₂ or ═CHR¹⁹, where R¹⁹ is(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl or C₆₋₁₀ aryl; or R¹⁰ is S(O)_(n)R¹⁶, where R¹⁶ is as definedabove and n=0, 1, or 2; wherein m=0, 1, or 2 and stereoisomers andpharmaceutically acceptable compositions thereof.

In a further embodiment of the present invention, the group R¹ as itappears in structure 3 may be H or (optionally substituted) C₁₋₄ alkyl,C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl,2-arylsubstituted ethynyl, arylsubstituted C₁₋₄ alkyl, arylsubstitutedC₂₋₄-alkenyl, arylsubstituted C₃₋₆ cycloalkyl, heterocycle;2-heterocycle-substituted ethynyl, heterocycle-substituted C₁₋₄ alkyl,heterocycle-substituted C₂₋₄-alkenyl, heterocycle-substituted C₃₋₆cycloalkyl;

R² group is OH or OR¹⁴, where R¹⁴ is HCO— or (optionally substituted)C₁₋₁₈ alkyl-CO— (except that R¹⁴ is not CH₃ when R¹, R², R³, R⁵, R⁶, R⁸,R⁹ and R¹⁰═H, R⁴═CH₃, and there are no double bonds present), C₂₋₁₈alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO—; orR¹⁴ is (optionally substituted) C₁₋₁₈ alkyl-X—CO—, C₂₋₁₈ alkenyl-X—CO—,C₂₋₁₈ alkynyl-X—CO—, C₆₋₁₀ aryl-X—CO— or heterocycle-X—CO—; or R¹⁴ istrimethylsilyl or triethylsilyl; or R¹⁴ is (optionally substituted) C₁₋₄alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkenyl, or C₂₋₄ alkynyl, or; or R¹⁴ isHO—SO₂— or a salt thereof or R² is NR¹⁵R¹⁶, where R¹⁵ is H, OH or(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl, C₆₋₁₀ aryl, heterocycle or R¹⁵ is OR¹⁷, where R¹⁷ is(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl, C₆₋₁₀ aryl, or heterocycle, C₁₋₁₈ alkyl-CO—, C₂₋₁₈ alkenyl-CO—,C₂₋₁₈ alkynyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO—; and where R¹⁶ is Hor (optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl,C₂₋₄ alkenyl, C₆₋₁₀ aryl, or heterocycle, or R¹⁶ is H—CO— or (optionallysubstituted) C₁₋₁₈ alkyl-CO—, C₂₋₁₈ alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₃₋₆cycloalkyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO—;

where X is O or NR¹⁰⁰, where R¹⁰⁰ is (optionally substituted) C₁₋₄alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl C₆₋₁₀ aryl orheterocycle; or

X is NOR¹¹⁰, where R¹¹⁰ is H or C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₆₋₁₂ aryl, or heteroaryl, any of which may beoptionally substituted; or

X is (H, H), (H, OH), (H, alkyl)₃), or (H, OCOR¹¹¹), where R¹¹¹ is C₁₋₆alkyl, C₃₋₈ cycloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₂ aryl, aralkyl,aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl orheteroaralkynyl, any of which may be optionally substituted; or

X is

where Y is —(CH₂)_(m)— where m is an integer of 0 to 3, or Y is—(CH₂)_(n)—Z—(CH₂)_(p)— where n is an integer of 0 to 2, p is an integerof 0 to 2 and Z is a heteroatom (optionally substituted) or Z is acarbon atom substituted with one or two C₁₋₆ alkyl groups;

R³ is H, halogen, cyano, azido or (optionally substituted) C₁₋₄ alkyl,C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, or heterocycle;

R⁴ is H or (optionally substituted) Me;

R⁶ is H, or (optionally substituted) C₁₋₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₈alkenyl, C₂₋₈ alkynyl, C₆₋₁₀ aryl or heterocycle;

R⁸ is H, halogen, azido, cyano, thiocyano, ═O, OH, OR¹⁶ (where R¹⁶ is asdefined above); or R⁵ is NH₂ or NR¹⁵R¹⁶ (where R¹⁵ and R¹⁶ are asdefined above); or R⁸ is (optionally substituted) C₁₋₁₈ alkyl, C₃₋₆cycloalkyl, C₂₋₁₈ alkynyl, C₂₋₁₈ alkenyl, C₆₋₁₀ aryl or heterocycle; orR⁸ is COOR¹⁸ where R¹⁸ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₆₋₁₀ aryl, or heterocycle; orR⁸ is S(O)_(n)R¹⁶, where R¹⁶ is as defined above and n=0, 1, or 2;

R⁹ is H, cyano, azido, halogen, thiocyano, OH, OR¹⁶, where R¹⁶ is asdescribed above, or R⁹ is (optionally substituted) C_(m) alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, 2-arylsubstitutedethynyl, arylsubstituted C₁₋₄ alkyl, arylsubstituted C₂₋₄-alkenyl,arylsubstituted C₃₋₆ cycloalkyl, heterocycle; 2-heterocycle-substitutedethynyl, heterocycle-substituted C₁₋₄ alkyl, heterocycle-substitutedC₂₋₄-alkenyl, heterocycle-substituted C₃₋₆ cycloalkyl;

R¹¹ is C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, C₃₋₈ cycloalkyl, C₆₋₁₂aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl,heteroaralkenyl or heteroaralkynyl any of which may by optionallysubstituted;

or

R¹¹ is

where R⁵⁰ and R⁶⁰ are each independently H; halogen; (R⁷⁰R⁸⁰N(O)_(r))—,where r is 0 or 1 and R⁷⁰ and R⁸⁰ are each independently H, C₁₋₆ alkyl,C₃₋₈ cycloalkyl, C₂₋₆ alkenyl or C₂₋₆ alkynyl, any of which may beoptionally substituted; substructure II,

where q is 0 or 1, Y is —(CH₂)_(m)— where m is an integer of 0 to 5, orY is —(CH₂)_(n)—Z—(CH₂)_(p)— where n is an integer of 0 to 2, p is aninteger of 0 to 2, and Z is a heteroatom (optionally substituted) andwhere the CH₂ groups may be optionally substituted; N-imidazolyl;—N-pyrrolyl-; HO—; CF₃SO₂—; C₁₋₆ alkyl-O—; C₁₋₆ perfluoroalkyl-O—; C₁₋₆alkyl-S—; C₁₋₆ alkyl-CH(OH)—; NC—; HCC—; C₆H₅CC—; 2′-furyl; 3′-furyl;2′-thiophenyl; 3′-thiophenyl; 2′-pyridyl; 3′-pyridyl; 4′-pyridyl;2′-thiazolyl; 2′-N-methylimidazolyl; 5′-pyrimidinyl; C₆H₅—; H₂C═CH—;C₁₋₆ alkyl; MeC(═CH₂)—; C₁₋₆ alkyl-CO; HCO; C₁₋₆ alkyl; C═NOR¹²⁰, orHC═NOR¹²⁰, where R¹²⁰ is H or C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₆₋₁₂ aryl, or heteroaryl, any of which may be optionallysubstituted; or R⁵⁰ and R⁶⁰ combine to form a ring

where W is CH₂, CH, NH, N, O, or S, and R⁹⁰ is H or C₁₋₆ alkyl; or R⁵⁰and R⁶⁰ combine to form a ring

where A and B are each independently H, F or C₁₋₆ alkyl or A and Bcombine to form ═O; or R⁵⁰ and R⁶⁰ combine to form a ring

where the CH₂ groups may be independently and optionally substituted;and R¹³ is one of hydrogen, thio-C₁₋₄-alkyl, thio-C₂₋₄-alkenyl, nitro,cyano, C₁₋₄ alkoxy, substituted C₁₋₄ alkoxy, C₂₋₄ alkenyloxy,aminocarbonyl, mono-C₁₋₄-alkylaminocarbonyl, di-C₁₋₄-alkylaminocarbonyl,sulfinyl, sulfonyl, thio, sulfonamido, C₂₋₄-alkynyloxy, optionallysubstituted C₆₋₁₀ aryloxy, optionally substituted C₆₋₁₀aryl-C₁₋₄-alkyloxy, an optionally substituted 1,3-dioxolan-4-one of anacetyl group, an optionally substituted 1,3-dioxan-4-one of an acetylgroup, an optionally substituted 1,3-oxathiolan-5-one of an acetylgroup, an optionally substituted 1,3-oxathian-5-one of an acetyl group,—O—C(O)—NR′R″, —C(O)—CH₂-J-G, —C(O)—CH₂—O-T, —C(O)—CH₂—O-E,—C(O)—CH₂-Q-G, —C(O)—CH₂-J′-Q-G, or —C(O)—CH₂-J′-Q-L, wherein

R′ and R″ independently represent hydrogen or optionally substitutedC₁₋₄ alkyl, or taken together with the nitrogen to which they areattached form a 3- to 6-membered heterocyclic ring;

J is one of S, SO or SO₂;

J′ is one of O, S, SO or SO₂;

Q is one of C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄ alkynyl;

G is one of C-attached hetero-C₅₋₁₀-aryl, optionally substituted C₆₋₁₀aryl, a quaternary ammonium salt of a nitrogen containinghetero-C₆₋₁₀-aryl group or a quaternary salt of an amino substitutedC₆₋₁₀ aryl group;

T is C-attached hetero-C₅₋₁₀-aryl or a quaternary ammonium salt of anitrogen containing hetero-C₅₋₁₀-aryl group;

E is optionally substituted C₆₋₁₀ aryl or a quaternary ammonium salt ofan amino substituted C₆₋₁₀ aryl group;

L is one of amino, amido, cyano, thiocyano, azido, nitro, hydroxy, halo,carboxyl, C₁₋₄ alkoxy, C₁₋₄ alkoxycarbonyl, C₁₋₄ alkanoyloxy, hydrogen,sulfate, thiosulfate, sulfonate, C₁₋₄ alkylthio, C₁₋₄ alkylsulfinyl,C₁₋₄ alkylsulfonyl or mercapto.

Specific Non-Limiting Examples Include the Compounds:

17β-Nitro-5α-androstan-3α-ol;3α-Hydroxy-3β-methyl-17β-nitro-5α-androstane;3α-Hydroxy-17β-thiomethyl-5α-androstane;11β-[4-(N,N-Dimethylamino)phenyl]-3α-hydroxy-3β-methyl-17β-nitro-5α□estrane;3β-Hydroxy-5α-androstan-17-oxime; 17β-Nitro-5α-androstan-3β-ol;17β-Nitro-21-nor-23-oxo-17α-cholan-3α-ol; 5′-Methylspiro-[androstan-17β,N-oxido-2′-pyrrolidine]-3α-ol; 5′-Methylspiro-[androstan-17β,2′-pyrrolidine]-3α-ol;1′-Formyl-5′-methylspiro-[androstan-17β,2′-pyrrolidine]-3α-ol;17β-Nitro-5α-androstan-3α-olbenzoylester;17β-Amino-5α-androstan-3α-olbenzoylester;17β-(Acetylamino)-5α-androstan-3α-ol benzoyl ester;17β-(Acetylamino)-5α-androstan-3α-ol;17β-[(1-Oxopropyl)-amino]-5α-androstan-3α-ol benzoyl ester;17β-[(1-Oxopropyl)-amino]-5α-androstan-3α-ol;17β-(N-Oxido-2-propaneimine)-5α-androstan-3α-ol;3α-Hydroxy-5α-androstan-17-thione; 3α-Hydroxy-5α-androstan-17β-thiol;3α-Hydroxy-5α-androstan-17β-methylsulfoxide (both diatereomers);3α-Hydroxy-5α-androstan-17β-methylsulfone;3α-Hydroxy-17-(2-furanyl)-5α□androst-16-ene;3α-Hydroxy-17β-(2-furanyl)-5α□androstane;11β-[4-(N,N-Dimethylamino)phenyl]-3α-hydroxy-3β-methyl-5α-17β-hydroxy-estranehydrochloride;11β-[4-(N,N-Dimethylamino)phenyl]-3α-hydroxy-3β-methyl-5α-estra-17-onehydrochloride;11β-[4-(N,N-Dimethylamino)phenyl]-3α-hydroxy-3β-methyl-5α-estrane-17-oxime;11β-[4-(N,N-Dimethyl-N-(oxy)amino)phenyl]-3α-hydroxy-3β-methyl-5α-estrane-17-oxime;3α-Amino-5α-androstan-17-one; 3α-Amino-17β-nitro-5αandrostanehydrochloride; 3α-Dimethylamino-17β-nitro-5α-androstane hydrochloride;3α-N-Hexylamino-17β-nitro-5α□androstane hydrochloride;3α-N-(3-Phenylpropyl)-17β-nitro-5α-androstane hydrochloride;3α-Acetamido-5α-17β-nitro-androstane.

Those compounds of the present invention, which bear an amino group, mayalso comprise a salt formed with the amine. Suitable pharmaceuticallyacceptable salts are known to those of ordinary skill in the artcomprise carboxylates, sulfates, phosphates and halides.

The compounds of the present invention may be administered by a varietyof methods. Thus, those products of the invention that are active by theoral route may be administered in solutions, suspensions, emulsions,tablets, including sublingual and intrabuccal tablets, soft gelatincapsules, including solutions used in soft gelatin capsules, aqueous oroil suspensions, emulsions, pills, lozenges, troches, tablets, syrups orelixirs and the like. Products of the invention active on parenteraladministration may be administered by depot injection, implantsincluding Silastic™ and biodegradable implants, skin patches, skincreams, or intramuscular and intravenous injections.

Compositions may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions and such compositionsmay contain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents. Tablets containing the active ingredient in admixture withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate granulating anddisintegrating agents, such as maize starch, or alginic acid; bindingagents, such as starch, gelatin or acacia; and lubricating agents, suchas magnesium stearate, stearic acid or talc. Tablets may be uncoated ormay be coated by known techniques to delay disintegration and adsorptionin the gastrointestinal tract and thereby provide a sustained actionover a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Ophthalmic formulations, as is known inthe art, will be adjusted for osmotic pressure.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oil suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water may beformulated from the active ingredients in admixture with a dispersing,suspending and/or wetting agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose disclosed above. Additional excipients, for example sweetening,flavoring and coloring agents, may also be present.

The pharmaceutical composition of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents include naturallyoccurring gums, such as gum acacia and gum tragacanth, naturallyoccurring phosphatides, such as soybean lecithin, esters or partialesters derived from fatty acids and hexitol anhydrides, such as sorbitanmono-oleate, and condensation products of these partial esters withethylene oxide, such as polyoxyethylenesorbitan mono-oleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent,such as a solution of 1,3-butanediol. Among the acceptable vehicles andsolvents that may be employed are water and Ringer's solution, anisotonic sodium chloride solution. In addition, sterile fixed oils mayconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid may likewisebe used in the preparation of injectables. Sterilization may beperformed by conventional methods known to those of ordinary skill inthe art such as, for example, by aseptic filtration, or irradiation.

Aqueous formulations (i.e oil-in-water emulsions, syrups, elixirs andinjectable preparations) may be formulated to achieve the pH of optimumstability. The determination of the optimum pH may be performed byconventional methods known to those of ordinary skill in the art.Suitable buffers may also be used to maintain the pH of the formulation.

The compounds of this invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable nonirritatingexcipient which is solid at ordinary temperatures but liquid at rectaltemperatures and will therefore melt in the rectum to release the drug.Non-limiting examples of such materials are cocoa butter andpolyethyleneglycols.

They may also be administered by intranasal, intraocular, intravaginal,and intrarectal routes including suppositories, insufflation, powdersand aerosol formulations.

Products of the invention which are preferably administered by thetopical route may be administered as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols.

The compounds according to the present invention may be administered toany warm-blooded mammal such as humans, domestic pets, and farm animals.Domestic pets include dogs, cats, etc. Farm animals include cows,horses, pigs, sheepgoats, etc.

The amount of active ingredient that may be combined with a carriermaterial to produce a single dosage form will vary depending upon thedisease treated, the mammalian species, and the particular mode ofadministration. A therapeutically effective amount may be determined byroutine experimentation and by analogy from the amounts used to treatthe same disease states with analogous steroid compounds. For example, aunit dose of the steroid may preferably contain between 0.1 milligramand 1 gram of the active ingredient. A more preferred unit dose isbetween 0.001 and 0.5 grams. It will be understood, however, that thespecific dose level for any particular patient will depend on a varietyof factors including the activity of the specific compound employed; theage, body weight, general health, sex and diet of the individual beingtreated the time and route of administration; the rate of excretion:other drugs which have previously been administered; and the severity ofthe particular disease undergoing therapy, as is well understood bythose of skill in the art.

Compounds of the invention may be made according to the proceduresoutlined in FIGS. 4, 5 and 6. Compounds of type A-2a,b where R═H or CH₃may be prepared from the requisite oxime (J. Med. Chem 2000, 3201-3204)A-1a,b by using N-bromosuccinimidein dioxane water followed by sodiumborohydride according to the method of Patchett et al. (Patchett, A. A.,Hoffman, F., Giarrusso, F. F., Schwam, H. and Arth, G. E., 1962, TheSynthesis of 17β-Amino-17α-(2′-carboxyethyl)androstane Lactams, J Org.Chem. 27, 3822). (see FIG. 4)

Compounds of type B-1-B-7 where prepared by treating3β-methyl-3α-trifluoroacetoxy-5α-androstan-17-one with benzylthiol inacetic acid/toluenesulfonic acid followed by ester cleavage withpotassium hydroxide in methanol according to the method of Swann et al.(Swann, D. A.; Turnbull, J. H.; TETRAB; Tetrahedron; EN; 24; 1968;1441-1444) The resulting bisbenzylthio B-2 is cleaved to thione B-3using sodium in liquid ammonia. Reduction of thione B-3 to provide the17β-thiol B-4 is accomplished with lithium tritertbutoxyaluminumhydride.Treatment of B-4 with methyl iodide accomplishes selective alkylationproviding B-5. Subsequent oxidation with metachloroperbenzoic acid at 0°C. in dichloromethane provided diastereomeric sulfoxides B-6 which wereseparated into their respective isomers using silica gel chromatography.Further oxidation with metachloroperbenzoic acid provided the sulfoneB-7. (see FIG. 5)

Compounds C1-C8 were prepared from11β-[4-(N,N-dimethylamino)phenyl]-17β-hydroxy-5α-estr-4-ene-3-one(Cleve, Arwed; Scheidges, Cornelius; Neef, Guenter; Ottow, Eckhard;Elger Walter; Beier, Sybille. Preparation of 11-β-aryl-4-estrenes asantiestagens and antiglucocorticoids, EP 404283). Compound C-1 wastreated with lithium metal in liquid ammonia to provide C-2 in moderateyield. Stereospecific addition of sodium trimethylsulfoxonium iodide toC-2 provided epoxide C-3 according to the method of Cook and colleagues.(Cook, C. E., R. C. Corley and M. E. Wall (1968). Steroids. LXXIX.Synthesis and reactions of oxiranes obtained from 3- and 17-ketosteroids. J. Org. Chem. 33(7): 2789-2793). Ring opening of C-3 withlithium aluminum hydride in tetrahydrofuran provided C-4 in excellentyield. Oxidation of C-4 with tetrapropylammonium perruthenate followedby treatment with hydroxylamine hydrochloride in pyridine gave oxime C-6in moderate yield. The aniline group in C-6 was protected as the N-oxideusing metachloroperbenzoic acid in dichloromethane. The final oxidationwas carried out using N-bromosuccinimide followed by sodium borohydridedioxane water according to the method of Patchett et al. (Patchett, A.A., Hoffman, F., Giarrusso, F. F., Schwam, H. and Arth, G. E., 1962, TheSynthesis of 17β-Amino-17α-(2′-carboxyethyl)androstane Lactams, J Org.Chem. 27, 3822). Deprotection of the N-oxide with ferric sulfate inwater provided the target molecule C-8 in moderate yield. (see FIG. 6)

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Synthetic Methods

Unless otherwise stated, reagent-grade chemicals and anhydrous solventswere obtained from commercial sources and were used without furtherpurification. All moisture and air-sensitive reactions and reagenttransfers were carried out under dry nitrogen or argon. Thin layerchromatography (TLC) was performed on EM Science precoated silica gel60F-254 plates. Compounds were normally visualized by UV light (254 nm)or p-anisaldehyde spray. Preparative column chromatography employed EMScience silica gel, 60 Å (230-400 mesh). Solutions were concentrated byuse of a rotary evaporator under water aspirator pressure at ambienttemperature. Melting points were taken on a Mel-Temp II and areuncorrected. Unless otherwise noted, ¹H NMR spectra were obtained at 300MHz on a Brucker Avance 300 spectrometer in CDCl3 as solvent withtetramethylsilane (TMS) as internal standard. Chemical shifts arereported in units of ppm downfield from TMS. Mass spectra were normallyobtained by electron impact at 70 eV on a Hewlett Packard 5989Ainstrument. Elemental analyses were performed by Atlantic Microlab Inc.,Atlanta, Ga.

Example 1 Synthesis of 17β-Nitro-5α-androstan-3α-ol3α-(t-butyldimethylsiloxy)-5α-androstan-17-oxime

Hydroxylamine hydrochloride (2 g, 29 mmol) was added to3α-(t-butyldimethylsiloxy)-5α-androstan-17-one (approx. 13 mmol) inanhydrous pyridine (200 mL). The solution was allowed to stir at roomtemperature for 14 h and concentrated under reduced pressure. Theresulting oil was purified on silica using medium pressure columnchromatography (hexane/EtOAc, 7:3 and3α-(t-butyldimethylsiloxy)-5α-androstan-17-oxime (4.85 g, 88%) wascollected and isolated as a shiny, white solid. ¹H-NMR (300 MHz, CDCl₃)δ 0.01, (s, 6H), 0.76 (s, 3H), 0.87 (s, 9H), 0.88 (s, 3H), 1.89-1.16 (m,22H), 2.55-2.46 (m, 2H), 3.94 (s, 1H).

3α-(t-butyldimethylsiloxy)-17β-nitro-5α-androstane

A solution of KHCO₃ (5.8 g, 57 mmol) in H₂O (60 mL) was added to asolution of NBS (5.1 g, 29 mmol) in dioxane (50 mL). The suspension wasallowed to stir at room temperature for 0.25 h and3α-(t-butyldimethylsiloxy)-5αandrostan-17-oxime (4.0 g, 9.5 mmol) indioxane (150 mL) was added in a dropwise manner. A pale green colorrapidly developed, and the reaction was allowed to stir at roomtemperature for 10 h. The solution was cooled to 0° C. and NaBH₄ (2.66g, 67 mmol) was added in portions. A large amount of gas evolution wasobserved and the reaction was allowed to stir overnight, graduallywarming to room temperature. The reaction was quenched with saturatedaqueous NH₄Cl (50 mL) and concentrated to ¼ its original volume. Theresulting suspension was partitioned between water (100 mL) and EtOAc(200 mL), and the layers were separated. The aqueous layer was thenextracted with EtOAc (3×100 mL) and organic extracts combined, washedwith brine (100 mL), dried (Na₂SO₄), and concentrated under reducedpressure. The resulting oil was purified on silica using medium pressurecolumn chromatography (hexane/EtOAc. 6:4). The solid product (3.36 g,84%) was collected and used without further purification. ¹H-NMR (300MHz, CDCl₃) δ 0.01 (s, 6H), 0.72 (s, 3H), 0.82 (s, 3H) 0.88 (s, 9H),2.03-0.89 (m, 23H), 2.67-2.41 (m, 1H), 3.94 (br s, 1H), 4.35 (dd, J=9.9Hz, 1H).

17β-nitro-5α-androstan-3α-ol

Aqueous HCl (1 M, 20 mL) was added to a suspension of3α-(t-butyldimethylsiloxy)-17β-nitro-5αandrostane (2.89 g, 6.6 mmol) inmethanol (100 mL) and was allowed to stir overnight at room temperature.The reaction mixture was concentrated under reduced pressure,partitioned between EtOAc (100 mL) and brine (50 mL), and the layerswere separated. The aqueous fraction was then extracted with EtOAc (2×50mL) and the organic extracts were combined, washed with saturatedaqueous NaHCO₃ (50 mL), brine (50 mL), dried over (Na₂SO₄), andconcentrated under reduced pressure. The resulting semisolid waspurified on silica using medium pressure column chromatography(hexane/EtOAc, 7:3) to provide 17β-nitro-5α-androstan-3α-ol (1.05 g,49%) as a pale yellow solid. ¹H-NMR (300 MHz, CDCl₃) δ 0.73 (s, 3H),0.79 (s, 3H), 2.05-0.86 (m, 23H), 2.70-2.52 (m, 1H), 4.05 (br s, 1H),4.37 (dd, J=9, 9 Hz, 1H).

Example 2 Synthesis of 3α-Hydroxy-3β-methyl-17β-nitro-5α-androstane3-Hydroxy-3β-methyl-17β-nitro-5α-androstane

KHCO₃ (0.55 g, 5.46 mmol) in H₂O (20 mL) was added to a well stirredsolution of NBS (0.50 g, 2.73 mmol) in dioxane (40 mL). The suspensionwas allowed to stir at room temperature for 0.25 h and a solution of3β-methyl-3α-hydroxy-5α-androstan-17-oxime (0.29 g, 0.91 mmol) indioxane (40 mL) was added. The blue solution was allowed to stir for 10h at room temperature and was then cooled to 0° C. NaBH₄ (0.25 g, 6.83mmol) was then added cautiously. After 3 h, H₂O (100 mL) was added andthe slurry was extracted with CH₂Cl₂ (3×100 mL). The organic extractswere combined, dried (MgSO₄) and concentrated under reduced pressure toprovide a white solid. The solid was purified on silica using mediumpressure column chromatography (petroleum ether/acetone, 8.5:1.5). Theresulting white solid was recrystallized from EtOAc/hexane to providewhite needles (0.10 g, 33%). mp. 211-213° C. ¹H-NMR (300 MHz, CDCl₃) δ0.72 (s, 3H), 0.76 (s, 3H), 0.79-1.75 (m, 25H), 2.03-2.08 (m, 2H),2.48-2.58 (m, 1H), 4.34-4.40 (dd, J=9.9 Hz, 1H). Elemental Anal. forC₂₀H₃₃NO₃ Calcd. C: 71.6, H: 9.91, N: 4.17. Found. C: 71.71, H: 9.97, N:4.21.

Ref for oxime. J. Med. Chem 2000, 3201-3204

Example 3 Synthesis of 3α-Hydroxy-17β-thiomethyl-5α-androstane17,17-Bisbenzylthio-5α-androstane-3α-ol

Toluenesulfonic acid (0.27 g, 1.43 mmol) was added to a well stirredsolution of benzyl mercaptan (2.41 g, 19.4 mmol) and 3α-trifluoroacetoxy-5α-androstan-17-one (3.0 g, 7.76 mmol) in glacial AcOH (30 mL).The reaction was allowed to stir at room temperature for 18 h and wasconcentrated under reduced pressure. The resulting oil was taken up in95% EtOH (140 mL) and 1N KOH (60 mL) was added. The slurry was thenheated at reflux for 4 h, cooled, and diluted with H₂O (200 mL). Themixture was extracted with CH₂Cl₂ (3×250 mL) and the organic fractionswere combined, dried (MgSO₄) and concentrated under reduced pressure.The resulting oil was purified on silica using medium pressure columnchromatography (petroleum ether/acetone, 9:1) to provide a white solid.The white solid was recrystallized from EtOAc/petroleum ether to providecolorless needles (3.76 g, 93%). mp. 184-185° C. ¹H-NMR (300 MHz, CDCl₃)δ 0.78 (s, 3H), 1.06 (s, 3H), 0.98-2.07 (m, 25H), 3.85-4.02 (m, 5H),7.23-7.35 (m, 10H).

Ref: Swann et al. Tetrahedron, Vol 24 1441-1444 1968

3α-hydroxy-5α-androstan-17-thione

17,17-Bisbenzylthio-5α-androstane-3α-ol (3.76 g, 7.22 mmol) in THF/Et₂O(40:100 mL) was added in a dropwise manner to a suspension of Na (3.32g, 144.4 mmol) in liquid NH₃ at −78° C. under N₂. The blue suspensionwas allowed to stir at −78° C. for 3 h. The reaction was then quenchedby the addition of saturated NH₄Cl (50 mL) and H₂O (150 mL). Theresulting biphasic mixture was extracted with CH₂Cl₂ (3×200 mL). Theextracts were combined, dried (MgSO₄), and concentrated to provide anorange semisolid. The semisolid was purified on silica using mediumpressure column chromatography (petroleum ether/acetone, 8:2). Theresulting solid was recrystallized from EtOAc/hexane to provide a salmoncolored solid (1.06 g, 48%). mp. 172-173° C. ¹H-NMR (300 MHz, CDCl₃) δ0.82 (s, 3H), 0.89 (s, 3H), 0.90-1.68 (m, 21H), 1.99-2.03 (m, 2H),2.59-2.69 (ddd, J=9, 9, 21 Hz, 1H), 2.90-3.00 (dd, J=9, 9 Hz, 1H), 4.06(m, 1H). Elemental Anal. for C₁₉H₃₀OS Calcd. C: 74.45, H: 9.87, S:10.46. Found. C: 74.33, H: 10.01, S: 10.47.

3α-hydroxy-5α-androstan-17β-thiol

A 1.0 M solution of lithium tri-t-butoxy aluminum hydride in THF (6.27mL, 6.27 mmol) was added in a dropwise manner to a solution of3α-hydroxy-5α-androstan-17-thione (0.64 g, 2.09 mmol) in THF (60 mL) at0° C. under N₂. The solution was allowed to stir for 2 h at 0° C. andthen another 2 h at room temperature. Saturated NaHCO₃ (75 mL) was addedand the slurry was extracted with CH₂Cl₂ (3×100 mL). The organics werecombined, dried (MgSO₄), and concentrated under reduced pressure. Theresulting white solid was recrystallized from heptane to provide a whitepowder (0.61 g, 95%). mp. 169-170° C. ¹H-NMR (300 MHz, CDCl₃) δ 0.70 (s,3H), 0.79 (s, 3H), 0.80-1.70 (m, 23H), 2.16-2.18 (m, 2H), 2.58-2.69 (dd,J=9, 18 Hz, 1H), 2.90-3.00 (dd, J=9, 9 Hz, 1H), 4.06 (m, 1H). ElementalAnal. for C₁₉H₃₂OS Calcd. C: 73.97, H: 10.45, S: 10.39. Found. C: 73.82,H: 10.73, S: 10.28.

3α-Hydroxy-17β-thiomethyl-5α-androstane

Methyl iodide (0.35 g, 2.48 mmol) was added to a well stirred suspensionof 3α-hydroxy-5α-androstan-17β-thiol (0.61 g, 1.98 mmol) and K₂CO₃ (1.38g, 10 mmol) in anhydrous DMF (50 mL). The suspension was allowed to stirat room temperature for 4 h and H₂O (100 mL) was added. The biphasicmixture was extracted with CH₂Cl₂ (3×150 mL) and the organics werecombined, dried (MgSO₄) and concentrated under reduced pressure. Theresulting solid was purified on silica using medium pressure columnchromatography (petroleum ether/acetone, 9:1) to afford a white solid.The solid was recrystallized from heptane to provide a white powder(0.19 g, 30%). mp. 151-153° C. ¹H-NMR (300 MHz, CDCl₃) δ 0.74 (s, 3H),0.79 (s, 3H), 0.80-1.55 (m, 23H), 1.63-1.69 (m, 1H), 2.11 (m, 4H),2.51-2.57 (dd, J=9, 9 Hz, 1H), 4.05 (m, 1H). Elemental Anal. forC₂₀H₃₄OS Calcd. C: 74.47, H: 10.62, S: 9.94. Found. C: 74.26, H: 10.81,S: 9.89.

Example 4 Synthesis of (+) and(−)3α-Hydroxy-5α-androstan-17β-methylsulfoxide (+) and(−)3α-Hydroxy-5α-androstan-17β-methylsulfoxide

MCPBA (0.16 g, 0.71 mmol, 77%) was added at 0° C. to a solution of3α-hydroxy-17β-thiomethyl-5α-androstane (0.19 g, 0.59 mmol) in CH₂Cl₂(100 mL). The solution was allowed to stir for 0.5 h at 0° C. andsaturated NaHCO₃ (50 mL) was added. The biphasic mixture was extractedwith CH₂Cl₂ (3×100 mL) and the organics were combined, dried (MgSO₄) andconcentrated under reduced pressure. The resulting semisolid waspurified on silica using medium pressure column chromatography(Et₂O/acetone, 9:1) to provide 0.050 g of each diatereomers and ≈0.075 gmixed fractions.

Top spot (11474-36t). The solid was recrystallized from EtOAc/hexane toprovide 3α-Hydroxy-5α-androstan-17β-methylsulfoxide as colorless needles(0.50 g, 50%). mp. 245-247° C. ¹H-NMR (500 MHz, CDCl₃) δ 0.79 (s, 3H),0.87 (s, 3H), 0.80-1.95 (m, 24H), 2.30-2.33 (dd, J=9, 9, Hz, 1H),2.37-2.44 (m, 1H), 2.49 (s, 3H), 4.05 (m, 1H). Elemental Anal. forC₂₀H₃₄O₂S Calcd. C: 70.96, H: 10.12, S: 9.47. Found. C: 70.86, H: 10.21,S: 9.44.

Bottom spot: The solid was recrystallized from EtOAc/hexane to provide3α-Hydroxy-5α-androstan-17β-methylsulfoxide as colorless needles (0.50g, 50%). mp. 251-253° C. ¹H-NMR (500 MHz, CDCl₃) δ 0.79 (s, 3H), 1.03(s, 3H), 0.98-2.41 (m, 24H), 2.48 (s, 3H), 2.59-2.63 (dd, J=9, 9 Hz,1H), 4.05 (m, 1H). Elemental Anal. for C₂₀H₃₄O₂S Calcd. C: 70.96, H:10.12, S: 9.47. Found. C: 70.77, H: 10.01, S: 9.40.

Example 5 Synthesis of 3α-Hydroxy-5α-androstan-17β-methylsulfoneSynthesis of 3α-Hydroxy-5α-androstan-17β-methylsulfone

MCPBA (0.10 g, 0.44 mmol) was added to a diatereomeric mixture of3α-hydroxy-5α-androstan-17β-methylsulfoxide (0.075 g, 0.22 mmol) inCH₂Cl₂ (50 mL). The solution was allowed to stir at room temperature for2 h, saturated NaHCO₃ was added (50 mL), and the biphasic mixture wasextracted with CH₂Cl₂ (3×100 mL). The organics were combined, dried(MgSO₄) and concentrated under reduced pressure. The resulting solid waspurified on silica using medium pressure column chromatography(acetone/petroleumether, 6:4). The product was collected andrecrystallied from EtOAc/heptane to provide3α-Hydroxy-5α-androstan-17β-methylsulfone as a white powder (0.061 g,78%). mp. 223-226° C. ¹H-NMR (300 MHz, CDCl₃) δ 0.79 (s, 3H), 1.04 (s,3H), 0.80-2.32 (m, 25H), 2.80 (s, 3H), 2.86-2.93 (dd, J=9.6, 9.6 Hz,1H), 4.13 (m, 1H). Elemental Anal. for C₂₀H₃₄O₃S.0.25H₂O Calcd. C:66.90, H: 9.68, S: 8.93. Found. C: 67.05, H: 9.62, S: 8.79.

Example 6 Synthesis of11β-[4-(N,N-Dimethylamino)phenyl]-3α-hydroxy-3β-methyl-5α-17β-nitro-estrane11β-[4-(N,N-dimethylamino)phenyl]-5α-17β-hydroxy-estr-3-one

Lithium metal (0.04 g, 6.35 mmol) was added to liquid NH₃ (40 mL) at−78° C. and allowed to stir for 15 min. Anhydrous THF (25 mL) was addedand then a solution of anhydrous THF (25 mL), t-butanol (0.30 mL), and11β-[4-(N,N-dimethylamino)phenyl]-5α-17β-hydroxy-estr-4-ene-3-one (1.0g, 2.54 mmol) was rapidly added. The blue solution was allowed to stirat −78° C. for 5 min and then quenched with solid NH₄Cl. Saturated NH₄Cl(150 mL) was then added and the biphasic mixture was extracted withEtOAc (3×150 mL). The organic extracts were combined, dried (MgSO₄), andconcentrated under reduced pressure. The resulting semisolid waspurified on silica using medium pressure column chromatography(petroleum ether/acetone, 8.5:1.5) to provide11β-[4-(N,N-dimethylamino)phenyl]-5α-17β-hydroxy-estr-3-one as acolorless semisolid (0.80 g, 80%). ¹H-NMR (300 MHz, CDCl₃) δ 0.52 (s,3H), 0.93-2.25 (m, 24H), 2.93 (s, 6H), 3.15 (m, 1H), 3.52 (dd, J=7.8,7.8 Hz, 1H), 6.41 (d, J=8.7 Hz, 2H), 7.23 (d, J=8.7 Hz, 2H).

11β-[4-(N,N-dimethylamino)phenyl]-spiro-3α-oxiranyl-11β-hydroxy-5α-estrane

A 60% dispersion of NaH in mineral oil (0.16 g, 4.05 mmol) was added toa solution of trimethylsulfoxonium iodide (0.89 g, 4.05 mmol) inanhydrous DMSO (100 mL) under N₂. The solution was allowed to stir atroom temperature for 2 h or until the evolution of gas ceased. Asolution of 11β-[4-(N,N-dimethylamino)phenyl]-5α-17β-hydroxy-estr-3-one(0.80 g, 2.02 mmol) in anhydrous DMSO (15 mL) was then added in adropwise manner. The suspension was allowed to stir at room temperaturefor 8 h and a saturated solution of NaCl (200 mL) was added. Thebiphasic mixture was extracted with CH₂Cl₂ (3×150 mL) and the organicextracts were combined, dried (MgSO₄) and concentrated under reducedpressure. The resulting oil was purified on silica using medium pressurecolumn chromatography (petroleum ether/acetone, 8:2) to provide11β-[4-(N,N-dimethylamino)phenyl]-spiro-3α-oxiranyl-17β-hydroxy-50α-estraneas a colorless oil (0.40 g, 48%). ¹H-NMR (300 MHz, CDCl₃) δ 0.45 (s,3H), 0.98-1.99 (m, 23H), 2.07 (d, J=13.2 Hz, 1H), 2.49 (d, J=4.8 Hz,1H), 2.53 (d, J=4.8 Hz, 1H), 2.87 (s, 6H), 3.20 (m, 1H), 3.47 (dd,J=7.8, 7.8 Hz, 1H), 6.58 (d, J=8.7 Hz, 2H), 7.19 (d, J=8.7 Hz, 2H).

11β-[4-(N,N-dimethylamino)phenyl]-1-hydroxy-3β-methyl-17β-hydroxy-5α-estrane

A solution of11β-[4-(N,N-dimethylamino)phenyl]-spiro-3α-oxiranyl-17β-hydroxy-5α-estrane(0.40 g, 0.98 mmol) in THF (15 mL) was added in a dropwise manner to awell stirred suspension of LiAlH₄ (0.11 g, 2.94 mmol) in anhydrous THF(50 mL) at 0° C. under N₂. The suspension was allowed to warm to roomtemperature and kept at room temperature for 8 h. Celite (1.0 g) wasadded followed by H₂O (0.15 mL) and 10% NaOH (0.15 mL). The viscoussuspension was filtered through a sintered glass funnel and the filtercake was washed with CH₂Cl₂ (2×50 mL). The resulting filtrate wasconcentrated under reduced pressure to provide a white solid. The solidwas taken up in MeOH (25 mL) and a 1.0 M solution of HCl in anhydrousether (1.0 mL) was added. The solution was concentrated under reducedpressure and the resulting solid was recrystallized from CH₃CN/petroleumether to provide11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-β-methyl-17β-hydroxy-5α-estranehydrochloride as a white solid (0.43 g, 94%). mp. 230-232° C. dec.¹H-NMR (300 MHz, CD₃OD) δ 0.19 (s, 3H), 0.85 (s, 3H), 0.62-1.80 (m,24H), 1.90-1.95 (d, J=13.2 Hz, 1H), 3.05 (s, 6H), 3.17 (m, 1H),3.27-3.32 (dd, J=8.7, 8.7 Hz, 1H), 7.23-7.26 (d, J=8.4 Hz, 2H),7.43-7.46 (d, J=8.4 Hz, 21-1). Elemental Anal. for C₂₇H₄₂ClNO₂.0.25H₂OCalcd. C: 71.65, H: 9.46, N: 3.09 Found. C: 71.90, H: 9.46, N: 3.25.

11β-[4-(N,N-dimethylamino)phenyl]-3β-hydroxy-3β-methyl-5α-estra-17-one

Tetrapropylammonium perruthenate (0.35 g, 0.10 mmol) was added to asolution of11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-β-methyl-17β-hydroxy-5α-estrane(0.38 g, 0.91 mmol), 4-methylmorpholine-N-oxide (0.21 g, 1.82 mmol), and4 Å powdered molecular sieves (0.05 g) in anhydrous CH₂Cl₂ (50 mL) atroom temperature under N₂. The black suspension was allowed to stir for10 h and tetrapropylammonium perruthenate (0.21 g, 0.60 mmol) was againadded. The black suspension was allowed to stir at room temperature foran additional 3 h and was filtered through a pad of Celite. The filtratewas concentrated under reduced pressure and the resulting black oil waspurified on silica using medium pressure column chromatography(petroleum ether/acetone, 8.5:1.5) to provide11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-β-methyl-5α-estra-17-onehydrochloride as a white solid (0.10 g, 28%). The hydrochloride salt wasprepared through the addition of 1.1 eq. of 1.0 M HCl in anhydrous Et₂Oto the amine in EtOAc. The resulting solid was filtered, washed withEt₂O and dried. mp. 222-225° C. dec. ¹H-NMR (300 MHz, CD₃OD) 0.67 (s,3H), 1.12 (s, 3H), 1.01-1.74 (m, 18H), 1.97 (m, 1H), 2.00-2.21 (m, 4H),2.46-2.57 (m, 1H), 3.36 (s, 6H), 3.57 (m, 1H), 7.54-7.57 (d, J=8.4 Hz,2H), 7.70-7.79 (d, J=8.4 Hz, 2H). Elemental Anal. for C₂₅H₄ClNO₂.0.25H₂OCalcd. C: 71.97, H: 9.05, N: 3.10 Found. C: 71.95, H: 9.09, N: 3.07.

11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-3β-methyl-5α-estrane-17-oxime

Hydroxylaminehydrochloride (0.038 g, 0.54 mmol) was added to a solutionof 11β-[4-(N,N-dimethylamino)phenyl]4-hydroxy-3β-methyl-R-estra-17-one(0.10 g, 0.24 mmol) in anhydrous pyridine (20 mL) at room temperatureunder N₂. The solution was allowed to stir at room temperature for 18 hand saturated NaHCO₃ (100 mL) was added. The biphasic mixture wasextracted with CH₂Cl₂ (3×100 mL) and the organic extracts were combined,dried (MgSO₄), and concentrated under reduced pressure to provide11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-3β-methyl-5α-estrane-17-oximeas a white solid (0.10 g, 98%). The product was used without furtherpurification. ¹H-NMR (300 MHz, CD₃OD) δ 0.55 (s, 3H), 0.98 (s, 3H),0.98-1.36 (m, 18H), 1.76-1.88 (m, 4H), 2.09 (m, 1H), 2.32 (m, 2H), 2.77(s, 6H), 3.20 (m, 1H), 6.58-6.61 (d, J=8.7 Hz, 2H), 7.15-7.18 (d, J=8.7Hz, 2H).

11β-[4-(N,N-dimethyl-N-(oxy)amino)phenyl]-3α-hydroxy-3β-methyl-5α-estrane-17-oxime

A solution of11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-3α-methyl-5α-estrane-17-oxime(0.10 g, 0.24 mmol) in CH₂Cl₂ (5 mL) was added in a dropwise manner to awell stirred solution of hexafluoroacetone (0.026 g, 0.12 mmol) and 30%H₂O₂ (0.053 mL, 0.47 mmol) in CH₂Cl₂ (15 mL). The solution was allowedto stir vigorously at room temperature for 3 h and H₂O (50 mL) wasadded. The biphasic mixture was extracted with H₂O (3×50 mL) and theaqueous extracts were combined and concentrated under reduced pressureto provide11β-[4-(N,N-dimethyl-N-(oxy)amino)phenyl]-3-hydroxy-3β-methyl-5α-estrane-17-oximeas a white solid (0.10 g, 99%). The solid was used without furtherpurification. ¹H-NMR (300 MHz, CD₃OD) δ 0.60 (s, 3H), 1.10 (s, 3H),0.63-1.66 (m, 18H), 1.87-2.05 (m, 4H), 2.23-2.28 (d, J=13.5 Hz, 1H),2.44 (m, 2H), 3.47 (m, 1H), 3.60 (s, 6H), 7.63-7.66 (d, J=8.7 Hz, 2H),7.85-7.88 (d, J=8.7 Hz, 2H).

11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-3β-methyl-17β-nitro-5α-estrane

KHCO₃ (0.20 g, 2.04 mmol) in H₂O (2 mL) was added to a well stirredsolution of NBS (0.18 g, 1.02 mmol) in dioxane (8 mL). The suspensionwas allowed to stir at room temperature for 0.25 h and a solution of11β-[4-(N,N-dimethyl-N-(oxy)amino)phenyl]-3α-hydroxy-3β-methyl-5α-estrane-17-oxime(0.15 g, 0.34 mmol) in dioxane (2 mL) and H₂O (2 mL) was added. The palegreen solution was allowed to stir at room temperature for 10 h and afreshly prepared aqueous solution of saturated FeSO₄ (50 mL) was added.The brown suspension was allowed to vigorously stir at room temperaturefor 0.25 h and was extracted with EtOAc (3×100 mL). The organic extractswere combined, washed with saturated FeSO₄ (3×100 mL), dried (MgSO₄),and concentrated under reduced pressure. The resulting brown oil wastaken up in THF/H₂O (20:2, 22 mL) and cooled to 0° C. NaBH₄ (0.09 g,2.38 mmol) was added and the solution was allowed to warm to roomtemperature and stir for 2 h. Water (50 mL) was added and the biphasicmixture was extracted with CH₂Cl₂ (3×100 mL). The organic extracts werecombined, dried (MgSO₄) and concentrated under reduced pressure. Theresulting oil was purified on silica using medium pressure columnchromatography (petroleum ether/acetone, 9:1) to provide11β-[4-(N,N-dimethylamino)phenyl]-3α-hydroxy-3β-methyl-5α-17β-nitro-estraneas a white solid (0.010 g, 7%). ¹H-NMR (500 MHz, CDCl₃) δ 0.48 (s, 3H),1.13 (s, 3H), 0.86-1.63 (m, 17H), 1.76-1.82 (m, 1H), 1.84-1.95 (m, 3H),1.99-2.07 (m, 1H), 2.36-2.39 (d, J=7.8 Hz, 1H), 2.42-2.51 (m, 1H), 2.99(s, 6H), 3.29 (m, 1H), 4.27 (dd, J=5.4, 5.4 Hz, 1H), 6.60-6.62 (d, J=5.1Hz, 2H), 7.19-7.21 (d, J=5.1 Hz, 2H). ¹³C-NMR (500 MHz, CDCl₃) 13.83,23.49, 24.34, 25.20, 31.42, 31.89, 31.92, 33.11, 37.63, 38.28, 38.52,38.78, 40.64, 42.16, 46.02, 46.21, 47.04, 50.89, 54.73, 69.61, 95.37,112.07, 130.26, 132.18, 148.16. HRMS: [M+H]⁺ for C₂₇H₄₀N₂O₃ Calcd.441.3117. Found 441.3116.

The biological activity of compounds detailed in this invention wereevaluated using in vitro and in vivo tests.

Radioligand Binding Assays

Tissue preparation. Rat brain cerebral cortex homogenates and thebinding assays were conducted essentially as described (Carter et al.,1997). Briefly, adult male Sprague-Dawley rats (Charles RiverLaboratories, Raleigh, N.C.) were killed by excess CO₂ asphyxiation,decapitated, and their brain rapidly removed and frozen on dry icebefore being stored −76° C. The frozen cortices were placed in 10volumes of an ice-cold 0.32 M sucrose solution and homogenized with aTeflon-glass homogenizer. The homogenate was centrifuged at 1500×g (10min at 4° C.). The supernatant was retained and centrifuged at 10,000×g(20 min at 4° C.), yielding the P2 pellet. The P2 pellet was resuspendedin an equal volume of a 50 mM Na+/K+ phosphate buffer containing 200 mMNaCl (pH 7.4) and centrifuged at 10,000×g (10 min at 4° C.). The pelletwas washed twice more, resuspended in 1/10 volume of buffer and storedat −76° C. until the time of assay.

[³⁵S]TBPS binding assays. These assays were carried out in 1.4 mLpolypropylene tubes (Matrix Technologies, Hudson, N.H.). In a finalvolume of 0.5 mL, each tube contained 100 μL, cortical membranesuspension (40 μg of protein, added last) 2 nM [³⁵S]TBPS (64-165Ci/mmol), 6.25 μM GABA and one of six different concentrations of testcompound added as 5 μL in 100% DMSO (1% final DMSO concentration). Thetest compounds were added to the assay tubes using a BiomekFX automatedliquid handler with 96-well head (Beckman-Coulter, Fullerton, Calif.).Nonspecific binding was determined in the presence of 200 μM picrotoxin.The assays were incubated for 2 h at RT before being terminated byvacuum filtration through 96-well GF/B glass filter plates using a96-well harvester (Brandel Scientific, Gaithersburg, Md.). Prior toharvesting, the filter plates were pre-soaked for 20 min in buffercontaining 0.15% BSA and 0.1% PEI. The radioactivity remaining on thefilter was determined in a TopCount 12-detector scintillation counter(Packard Instruments, Meriden, Conn.) using 20 μl of MicroScint20(Packard Instruments) per well and standard liquid scintillationcounting techniques.

[³H]Flunitrazepam binding assay. This assay was conducted as describedfor the [³⁵S]TBPS assay except that the assays contained 20 μg tissuehomogenate, 1.0 μM GABA and 1.0 nM [³H]flunitrazepam (74.1-85 Ci/mmol).Clonazepam (1.0 μM) was used to determine nonspecific binding.

Data analysis. The IC₅₀ or EC₅₀ and E_(max) values for allopregnanoloneand the test compounds were calculated from a three-parameter logisticequation fit to the binding data using Prism (version 3, GraphPadSoftware, San Diego, Calif.) and are reported in Table 2 below.

TABLE 2 Lead Structures: Highly Potent and Efficacious NeuroactiveSteroids [³H]Flunitrazepam [³⁵S]TBPS Compound Compound EC₅₀ (nM) E_(max)(%) IC₅₀ (nM) Allopregnanolone

 26 ± 9.9 100% 12.6 ± 1.7  A2-A

 24 ± 2.5 103%   13 ± 0.13 A2-B

33.5 ± 4.9  99.1%  17.9 ± 6.19 B-5

75.9 ± 12.4 131.7%     32 ± 5.19 C-8

39.5 ± 20.6 135% 9.79 ± 1.93

The E_(max) values are for pregnanolone and the test compounds arepresented as the percentage of E_(max) determined for allopregnanolone.The average enhancement of [³H]flunitrazepam measured in the presence ofallopregnanolone was approximately 60%. In cases where limitedinhibition of [³⁵S]TBPS or enhancement of [³H]flunitrazepam occurred atthe top concentration of 10,000 nM, the IC₅₀ or EC₅₀ values are givenas >10,000 nM. The data for the active compounds are reported as themean±SD from at least three independent experiments.

Testing in Mouse Models

Male NIH Swiss mice (25-30 g) were housed two per cage. Animals werekept in a vivarium (temperature 22-26° C.; humidity 40-50%) with anartificial 12-h light/dark cycle and free access to food and water.Animals were allowed to acclimate to the vivarium for at least 5 days.The experiments were performed during the light phase of the light/darkcycle (between 9:30 AM and 3:30 PM), after at least a 30-min period ofacclimation to the experimental room. Animals were maintained infacilities fully accredited by the Association for Assessment andAccreditation of Laboratory Animal Care, and experiments were performedunder protocols approved by the Animal Care and Use Committee of theNational Institute of Neurological Disorders and Stroke in strictcompliance with the Guide for the Care and Use of Laboratory Animals ofthe National Research Council (National Academy Press, Washington, D.C.:(http://www.nap.edu/readingroom/books/labrats/).

Solutions of the steroids were made fresh daily in 40%hydroxypropyl-β-cyclodextrin in sterile 0.9% saline. Further dilutionswere made using sterile saline. The steroids were injectedintraperitoneally. The convulsant agent pentylenetetrazol (PTZ;Sigma-Aldrich) was dissolved in saline immediately before use. All drugsolutions were administered in a volume equaling 0.01 mL/g of theanimal's body weight.

The PTZ seizure test was carried out as described (Kokate et al., 1994).In brief, mice were injected subcutaneously with PTZ (80 mg/kg) 15 minafter injection of the test steroid and were observed for a 30-minperiod. Mice failing to show clonic seizures lasting longer than 5 swere scored as protected. To construct dose-response curves, steroidswere tested at several doses spanning the dose producing 50% protection(ED₅₀). Six to eight mice were tested at each dose. ED₅₀ values andcorresponding 95% confidence limit were determined by log-probitanalysis.

The 6-Hz seizure test was carried out 15 min after injection of the teststeroid as described (Kaminski et al., 2004). In brief, 3-s cornealstimulation (200 μs-duration, 32-mA monopolar rectangular pulses at 6Hz) was delivered by a constant current device (ECT Unit 5780; UgoBasile, Comerio, Italy). Ocular anesthetic (0.5% tetracaine) was appliedto the corneas 15 min before stimulation. In response to stimulation,the animals exhibited a “stunned” posture associated with rearing andautomatic movements that lasted from 60-120 s in untreated animals.Animals resumed their normal exploratory behavior after the seizure. Theexperimental endpoint was protection against the seizure. An animal wasconsidered to be protected if it resumed its normal exploratory behaviorwithin 10 s of stimulation. The statistical comparisons were the same asdescribed for the PTZ test.

Anxiolytic activity was assessed with the elevated zero-maze asdescribed (Kaminski et al., 2006). The maze (Hamilton-Kinder, Poway,Calif.) was positioned in the center of a room under dim lighting. Eachmouse was individually removed from its home cage and placed just insidea closed arm. Five-min test sessions were video-recorded with atripod-mounted camcorder. Percent time in open areas and number ofentries into open areas were scored from the taped records usingObserver 3.0 software (Noldus, Wageningen, The Netherlands). An animalwas considered to have entered an open area if all four paws had leftthe closed areas. Open-area time was considered terminated once a singlepaw was placed back into the closed area. Increases in these measuresreflect anxiolytic activity. Dose-response data were analyzed by one-wayanalysis of variance. Differences from control (vehicle-treatment)values for individual doses were identified by post-hoc comparisonsusing the Dunnett's test.

The series of GABA_(A) receptor ligands described herein display bindingcharacteristics on par with the endogenous neurosteroidallopregnanolone.

It will now be apparent to those skilled in the art that numerousmodifications and variations of the present invention are possible inlight of the above teachings. It is therefore to be understood that,within the scope of the appended claims, the invention may be practicedotherwise than as specifically described herein without departing fromthe spirit and the scope of the present invention.

REFERENCES U.S. Patent Documents

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1-2. (canceled)
 3. A compound of structure 3, wherein:

R¹ is H or (optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, 2-arylsubstituted ethynyl,arylsubstituted C₁₋₄ alkyl, arylsubstituted C₂₋₄-alkenyl,arylsubstituted C₃₋₆ cycloalkyl, heterocycle, 2-heterocycle-substitutedethynyl, heterocycle-substituted C₁₋₄ alkyl, heterocycle-substitutedC₂₋₄-alkenyl, or heterocycle-substituted C₃₋₆ cycloalkyl; R² group is OHor OR¹⁴, where R¹⁴ is HCO— or (optionally substituted) C₁₋₁₈ alkyl-CO—(except that R¹⁴ is not CH₃ when R¹, R², R³, R⁵, R⁶, R⁸, R⁹ and R¹⁰═H,R⁴═CH₃, and there are no double bonds present), C₂₋₁₈ alkenyl-CO—, C₂₋₁₈alkynyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO—; or R¹⁴ is (optionallysubstituted) C₁₋₁₈ alkyl-X—CO—, C₂₋₁₈ alkenyl-X—CO—, C₂₋₁₈alkynyl-X—CO—, C₆₋₁₀ aryl-X—CO— or heterocycle-X—CO—; or R¹⁴ istrimethylsilyl or triethylsilyl; or R¹⁴ is (optionally substituted) C₁₋₄alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkenyl, or C₂₋₄ alkynyl, or; or R¹⁴ isHO—SO₂— or a salt thereof or R² is NR¹⁵R¹⁶, where R¹⁵ is H, OH or(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl, C₆₋₁₀ aryl, heterocycle or R¹⁵ is OR¹⁷, where R¹⁷ is(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl, C₆₋₁₀ aryl, or heterocycle, C₁₋₁₈ alkyl-CO—, C₂₋₁₈ alkenyl-CO—,C₂₋₁₈ alkynyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO—; and where R¹⁶ is Hor (optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl,C₂₋₄ alkenyl, C₆₋₁₀ aryl, or heterocycle, or R¹⁶ is H—CO— or (optionallysubstituted) C₁₋₁₈ alkyl-CO—, C₂₋₁₈ alkenyl-CO—, C₂₋₁₈ alkynyl-CO—, C₃₋₆cycloalkyl-CO—, C₆₋₁₀ aryl-CO— or heterocycle-CO—; where X is O orNR¹⁰⁰, where R¹⁰⁰ is (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl C₆₋₁₀ aryl or heterocycle; or Xis NOR¹¹⁰, where R¹¹⁰ is H or C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₆₋₁₂ aryl, or heteroaryl, any of which may be optionallysubstituted; or X is (H, H), (H, OH), (H, OSi(C₁₋₆ alkyl)₃), or (H,OCOR¹¹¹), where R¹¹¹ is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₆₋₁₂ an, aralkyl, aralkenyl, aralkynyl, heteroaryl,heteroaralkyl, heteroaralkenyl or heteroaralkynyl, any of which may beoptionally substituted; or X is

where Y is —(CH₂)_(m)— where m is an integer of 0 to 3, or Y is—(CH₂)_(n)—Z—(CH₂)_(p)— where n is an integer of 0 to 2, p is an integerof 0 to 2 and Z is a heteroatom (optionally substituted) or Z is acarbon atom substituted with one or two C₁₋₆ alkyl groups; R³ is H,halogen, cyano, azido or (optionally substituted) C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl, C₆₋₁₀ aryl, or heterocycle; R⁴is H or (optionally substituted) Me; R⁶ is H, or (optionallysubstituted) C₁₋₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl,C₆₋₁₀ aryl or heterocycle; R⁸ is H, halogen, azido, cyano, thiocyano,═O, OH, OR¹⁶ (where R¹⁶ is as defined above); or R⁵ is NH₂ or NR¹⁵R¹⁶(where R¹⁵ and R¹⁶ are as defined above); or R⁸ is (optionallysubstituted) C₁₋₁₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₁₈ alkynyl, C₂₋₁₈ alkenyl,C₆₋₁₀ aryl or heterocycle; or R⁸ is COOR¹⁸ where R¹⁸ is (optionallysubstituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl,C₆₋₁₀ aryl, or heterocycle; or R⁸ is S(O)_(n)R¹⁶, where R¹⁶ is asdefined above and n=0, 1, or 2; R⁹ is H, cyano, azido, halogen,thiocyano, OH, OR¹⁶, where R¹⁶ is as described above, or R⁹ is(optionally substituted) C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₂₋₄ alkynyl, C₂₋₄alkenyl, C₆₋₁₀ aryl, 2-arylsubstituted ethynyl, arylsubstituted C₁₋₄alkyl, arylsubstituted arylsubstituted C₃₋₆ cycloalkyl, heterocycle;2-heterocycle-substituted ethynyl, heterocycle-substituted C₁₋₄ alkyl,heterocycle-substituted C₂₋₄-alkenyl, heterocycle-substituted C₃₋₆cycloalkyl; R¹¹ is C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, C₃₋₈cycloalkyl, C₆₋₁₂ aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl,heteroaralkyl, heteroaralkenyl or heteroaralkynyl any of which may byoptionally substituted; or R¹¹ is

where R⁵⁰ and R⁶⁰ are each independently H; halogen; (R⁷⁰R⁸⁰N(O)_(r))—,where r is 0 or 1 and R⁷⁰ and R⁸⁰ are each independently H, C₁₋₆ alkyl,C₃₋₈ cycloalkyl, C₂₋₆ alkenyl or C₂₋₆ alkynyl, any of which may beoptionally substituted; substructure II,

where q is 0 or 1, Y is —(CH₂)_(m)— where m is an integer of 0 to 5, orY is —(CH₂)_(n)—Z—(CH₂)_(p)— where n is an integer of 0 to 2, p is aninteger of 0 to 2, and Z is a heteratom (optionally substituted) andwhere the CH₂ groups may be optionally substituted; N-imidazolyl;—N-pyrrolyl-; HO—; CF₃SO₂O—; C₁₋₆ alkyl-O—; C₁₋₆ perfluoroalkyl-O—; C₁₋₆alkyl-S—; C₁₋₆ alkyl-CH(OH)—; NC—; HCC—; C₆H₅CC—; 2′-furyl; 3′-furyl;2′-thiophenyl; 3′-thiophenyl; 2′-pyridyl; 3′-pyridyl; 4′-pyridyl;2′-thiazolyl; 2′-N-methylimidazolyl; 5′-pyrimidinyl; C₆H₅—; H₂C═CH—;C₁₋₆ alkyl; MeC(═CH₂)—; C₁₋₆ alkyl-CO; HCO; C₁₋₆ alkyl; C═NOR¹²⁰, orHC═NOR¹²⁰, where R¹²⁰ is H or C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₆₋₁₂ aryl, or heteroaryl, any of which may be optionallysubstituted; or R⁵⁰ and R⁶⁰ combine to form a ring

where W is CH₂, CH, NH, N, O, or S, and R⁹⁰ is H or C₁₋₆ alkyl; or R⁵⁰and R⁶⁰ combine to form a ring

where A and B are each independently H, F or C₁₋₆ alkyl or A and Bcombine to form ═O; or R⁵⁰ and R⁶⁰ combine to form a ring

where the CH₂ groups may be independently and optionally substituted;and R¹³ is one of hydrogen, thio-C₁₋₄-alkyl, thio-C₂₋₄-alkenyl, cyano,aminocarbonyl, mono-C₁₋₄-alkylaminocarbonyl, di-C₁₋₄-alkylaminocarbonyl,sulfinyl, sulfonyl, thio, sulfonamido, C₂₋₄-alkynyloxy, optionallysubstituted C₆₋₁₀ aryloxy, optionally substituted C₆₋₁₀aryl-C₁₋₄-alkyloxy, an optionally substituted 1,3-dioxolan-4-one of anacetyl group, an optionally substituted 1,3-dioxan-4-one of an acetylgroup, an optionally substituted 1,3-oxathiolan-5-one of an acetylgroup, an optionally substituted 1,3-oxathian-5-one of an acetyl group,—O—C(O)—NR′R″, —C(O)—CH₂-J-G, —C(O)—CH₂—O-T, —C(O)—CH₂—O-E,—C(O)—CH₂-Q-G, —C(O)—CH₂-J′-Q-G, or —C(O)—CH₂-J′-Q-L, wherein R′ and R″independently represent hydrogen or optionally substituted C₁₋₄ alkyl,or taken together with the nitrogen to which they are attached form a 3-to 6-membered heterocyclic ring; J is one of S, SO or SO₂; J′ is one ofO, S, SO or SO₂; Q is one of C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄ alkynyl; Gis one of C-attached hetero-C₅₋₁₀-aryl, optionally substituted C₆₋₁₀aryl, a quaternary ammonium salt of a nitrogen containinghetero-C₅₋₁₀-aryl group or a quaternary salt of an amino substitutedC₆₋₁₀ aryl group; T is C-attached hetero-C₅₋₁₀-aryl or a quaternaryammonium salt of a nitrogen containing hetero-C₅₋₁₀-aryl group; E isoptionally substituted C₆₋₁₀ aryl or a quaternary ammonium salt of anamino substituted C₆₋₁₀ aryl group; L is one of amino, amido, cyano,thiocyano, azido, nitro, hydroxy, halo, carboxyl, C₁₋₄ alkoxy, C₁₋₄alkoxycarbonyl, C₁₋₄ alkanoyloxy, hydrogen, sulfate, thiosulfate,sulfonate, C₁₋₄ alkylthio, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl ormercapto. and stereoisomers and pharmaceutically acceptable compositionsthereof. 4-10. (canceled)
 11. The method for treatment of a conditionselected from the group consisting of convulsions, epilepsy, depression,drug and alcohol abuse, anxiety, memory problems, and neural systemdamage in humans or animals, comprising: administering, to a human oranimal in need thereof, a pharmaceutical composition comprising aneffective amount of the compound of claim 3 and a pharmaceuticallyacceptable carrier.
 12. A pharmaceutical composition, comprising: thecompound according to claim 3, and a pharmaceutically acceptablecarrier.